To study the mechanism on atherosclerosis, low density lipoproteins (LDL) were separated from plasma of patients with atherosclerotic diseases and from the extracts of aortas obtained from 3 cases died by cerebral thrombosis. In addition, to clarify the mechanism of lipid accumulation due to cholesterol loading on arteries, plasma LDL and LDL in aortic wall were isolated by ultracentrifugation.
LDL lipid constituents, LDL molecular sizes and the effects of infused LDL on the arterial endothelial cells were studied.
1) LDL lipid separated from plasma of the patients with atherosclerotic diseases changed to peroxidized lipid such as hydroperoxidized cholestery linoleate (HPO-CL) with high rate.
2) The production of HPO-CL did not relate directly to plasma cholesterol level and cholesteryl ester levels.
3) Although the substance with the same Rf value as HPO-CL was recognized in total extracts obtained from human aortic atheroma, the substance was the reduced lipid of HPO-CL.
4) LDL molecular size due to negative stain became to large particle week by week after 1% cholesterol feeding in rabbit but HPO-CL was not stained in the LDL lipid isolated from plasma and aortic total extracts.
5) Arterial endothelial cell injuries and fixation of blood corpuscles on arterial endothelial cell were observed by the infusion into rabbit auricular vein of HPO-CL LDL or large molecular sized LDL.
Conclusion
Peroxidized LDL relates deeply to the mechanism of atherosclerosis fromed for long period such as human atherosclerotic diseases, while peroxidized LDL does relate to the mechanism of acute lipid accumulation formed for short period such as cholesterol feeding rabbit but relates deeply to large molecular sized LDLs.

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Lipid-soluble cholesteryl ester core aldehydes (aldehydes bound to the cholesterol ring) were identified among the products of copper-catalyzed peroxidation of human low density. lipoprotein (LDL). The core aldehydes were mainly the C₉, C₈ and C₅ oxoalkanoyl esters of cholesterol and 7-ketocholesterol with smaller amounts of C₄, C₆, C₇ and C₁₀ homologues.
The core aldehydes produced by tert-butyl hydroperoxide/Fe⁺⁺ peroxidation of cholestery linoleate were identified as mainly 9-oxononanoates of cholesterol and 7-ketocholesterol. Peroxidation of cholesteryl arachidonate yielded 5-oxovalerates of cholesterol and 7-ketocholesterol as the main products. Cholesteryl palmitate and oleate did not yield core aldehydes in the present peroxidation system.
Phosphatidylcholine core aldehydes were also identified among the products of copper-catalyzed peroxidation of LDL.
The implications of core aldehydes in atherogenesis and the role of lysosomes of macrophages in generation of core aldehydes in vivo are discussed.

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When serum low-density lipoprotein (LDL) was peroxidized and incubated with an epinephrine-FeCl₃ mixture in the presence of spin trap 5, 5-dimethyl-l-pyrrolin-N-oxide (DMPO), an electron spin resonance spectrum indicative of the spin adduct of DMPO and hydroxyl radical (·OH) was detected. The amount of ·OH increased with an increase in the content of lipid hydroperoxides in the LDL. These results indicate that ·OH was generated from lipid hydroperoxides in the LDL and that cytotoxicity of peroxidized LDL is at least partly attributable to the hazardous effect of the ·OH thus generated. Since lipid hydroperoxides are rather stable and can be transferred from one site to another via LDL, we consider that lipid hydroperoxides are a propagator of radical-mediated injury to tissues and organs.
Serum LDL comprises distinct subclasses. We purified these three subfrations of LDL, LDL-1, LDL-2, and LDL-3, to homogeneity from hyper-cholesterolemic rabbits. On the basis of size and density distribution, LDL-1 corresponded to intermediate density lipoprotein obtained by the standard sequential floatation method. When the LDL subfractions were incubated in the presence of Cu²⁺, the lipid peroxide level and the mobility in agarose gel electrophoresis were increased in the following order: LDL-1>LDL-2>LDL-3>normal LDL, indicating LDL-1 to be the most susceptible to lipid peroxidation. The modified LDL samples were taken up by rat peritoneal macrophages in the same order as listed above.
Taking into account the facts that lipid hydroperoxides generate ·OH and LDL-1 is the most susceptible to lipid peroxidation, this subtraction seems to be more atherogenic than the other LDL subfractions.

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In this study we established a murine monoclonal antibody that recognizes oxidized LDL using homogenates of human atheromatous plaque as immunogen. This antibody, FOH1a/DLH3, reacted with oxidized LDL but did not with either native, acetylated or malondialdehyde-treated LDL on ELISA. The antibody cross-reacted with oxidized HDL, suggesting that particular sequences of the apoB are not essential for specificity of the antigen recognition by the antibody. Immunohistochemical analysis of thin paraffin-sections from human colonary arteries showed that the foam cells derived from macrophages in atherosclerotic lesion were stained conspicuously with this antibody. Other compositions in the lesion including cellular debris in necrotic cores, swollen collagen fibers and endothelial cells were moderately stained. The epitope of this antibody was characterized by a model antigen generating system using ferrous ion-induced peroxidation of lipids. When the total lipid fraction extracted from LDL was treated with the ferrous ion-induced peroxidation system, the reaction mixture was recognized by the antibody. Antigenic product(s) was produced from phosphaidylcholine (PC) but not from the other lipids in the ferrous ion-induced peroxidation system. To detect a complex of an antigenic product with a polypeptide, reaction mixture of PC oxidized in the presence of a synthetic peptide was added to a microtiter well precoated with the monoclonal antibody FOH1a/DLH3. After washing, the peptide remained in the well was detected with a rabbit antiserum against the peptide, while no reactivity was obtained when the peptide alone was added to the well. Binding of the complex of oxidized PC and the peptide to FOH1a/DLH3 was competed by oxidized PC that was genarated in the absence of any polypeptide. We conclude that oxidized phospholipid product(s) is the epitope of the monoclonal antibody which forms complexes with polypeptides, and that the antigenic materials are detected in foam cells in atherosclerotic lesion.

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When mouse macrophages were incubated with oxidized LDL (Ox-LDL) together with discoidal complexes of apolipoprotein A-I and dimyristoyl-phosphatidylcholine (DMPC/apoA-I), cholesteryl ester (CE) accumulation was strongly inhibited. Endocytic degradation of Ox-LDL by macrophages was completely inhibited by the presence of DMPC/apoA-I, indicaing interaction of DMPC/apoA-I with Ox-LDL might occur in the medium. To characterize the interaction of DMPC/apoA-I with Ox-LDL, Ox-LDL was incubated with DMPC/apoA-I in a cell-free system and re-isolated on Sephacryl S-400 gel filtration chromatography. Physico-chemical analysis of re-isolated Ox-LDL showed a 2-fold increase in phospholipids and a 10% increase in the protein moiety. The electrophoretic mobility of re-isolated Ox-LDL was significantly reduced as compared with control Ox-LDL. Cellular degradation of re-isolated Ox-LDL was reduced by<70%, suggesting a significant reduction in its ligand activity for the scavenger receptor. Incubation of Ox-LDL with free apoA-I led to apo-A incorporation into Ox-LDL, however, it did not alter the ligand activity. Thus, it was concluded that transfer of DMPC from DMPC/apoA-I to Ox-LDL induced altered recognition by the scavenger receptor. Because discoidal HDL particles similar to DMPC/apoA-I virtually are known to exist in the interstitial fluid, we propose here a neutalizing effect of discoidal HDL on Ox-LDL being a new mechanism for anti-atherogenic function of HDL in vivo.

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Several line of evidence indicate that the oxidative modification of low density lipoprotein (LDL) may provide an important link between plasma LDL and the genesis of atherosclerotic lesion. Ascorbic acid is an important water soluble, chain breaking antioxidant in human. The aim of the present study was to examine the effect of physiologic level of ascorbic acid (10μg/ml) on Cu²⁺ induced oxidative modification of LDL. Ascorbic acid had an inhibitary effect on the oxidative modification of LDL for 6h, as evidenced by the electrophoretic mobility and concentration of polyunsaturated fatty acid. Also, ascorbic acid aided the provisin of cholesterol to human lymphocyte through LDL recepter mechanism by LDL. These findings indicate that ascorbic acid in physiologic concentration should inhibit the oxidative modification of LDL in vivo.

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Antioxidants may be useful in the prevention of coronary heart disease by inhibiting low density lipoprotein (LDL) oxidation, which is believed to play an important role in atherogenesis. The structures of the 6'- and 7'-hydrxy-metabolites of doxazosin (6'- and 7'-OH doxazosin), and α-1 adrenergic blocking antihypertensive agent, suggests that they may have antioxidant properties. Micromolar concentration of 6'- and 7'-OH doxazosin, but not doxazosin itself, inhibited copper-mediated oxidative modification of LDL in a dose-dependent fashion, similar to that observed with the lipophilic antioxidant, probucol. LDL modified in the presence of these metabolites was not taken up and degraded by macrophages to the same extent as LDL oxidized in their absence. Whereas probucol, like vitamin E, sequesters with LDL, ³H-labeled 6'- and 7'-OH doxazosin did not comigrate with lipoprotein on FPLC, but were associated with albumin and occurred free in solution. Thus these metabolites of doxazosin are likely to exert their antioxidant effect in the aqueous milieu of the lipoprotein, similar to vitamin C, and may be useful for the prevention of atherosclerosis in hypertentive individuals.

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Although the hydroxymethylglutaryl coenzyme A reductase inhibitor is a powerful therapeutic agent in the treatment of hypercholesterolemia, an escape phenomenon has been reported. We sought to determine the effects of simvastatin in patients associated with the escape phenomenon following pravastatin therapy. Twenty-one patients with hypercholesterolemia (mean age:58;age range:25 to 75;seven men, 14 women) were studied. All patients had been given pravastatin (10mg/day) for at least 12 weeks. After the occurrence of the escape phenomenon, pravastatin was replaced by simvastatin (5mg/day). Serum lipids were measured periodically. Although total serum cholesterol levels showed significant decreases following pravastatin therapy (from 281±4mg/dl to 224±6mg/dl), such levels increased to 255±6mg/dl after 12 to 116 weeks. By replacing pravastatin with simvastatin, total serum cholesterol levels fell to 220±5mg/dl after 12 weeks and remained low (222±8mg/dl, n=13) even after 32 weeks. However, the total serum cholesterol levels tended to increase after 40 weeks. The serum LDL-cholesterol levels revealed changes that paralleled those of the total cholesterol levels, while serum HDL-cholesterol and the triglyceride levels did not reveal significant changes. These results suggest that simvastatin is a useful therapeutic alternative when the escape phenomenon occurs involving the use of pravastatin, although a similar phenomenon may occur even with simvastatin following long-term administration.

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We investigated the effects of simvastatin and pravastatin, which are inhibitors of HMG-CoA reductase, on serum lipoproteins by administrating pravastatin (P) and simvastatin (S) to the same hypercholesterolemic patients. We then analyzed the mechanism by which simvastatin increases HDL-C. Ten mg of P was administrated daily to 28 outpatients with hypercholesterolemia for more than 16 weeks. These patients were then given daily 5mg doses of S for further 16 weeks in exchange for P. At the end of each administration period (at the time of cessation of the drug for pravastatin and at week 16 for simvastatin), sera were obtained to determine serum lipoproteins and apolipoproteins as well as blood chemistry and hematology. Total cholesterol (TC or C), low density lipoprotein (LDL)-C, and LDL-triglyceride (TG) were significantly decreased by treatment with P and S. S decreased TC and LDL-C significantly greater than P did, respectively. S also significantly dereased very low-density lipoprotein (VLDL)-TG and significantly increased HDL-C (determined by a precipitation method using phosphotungstic acid-dextran sulfate). Though apolipoproteins (apo) B, C-II, and E were significantly reduced by both statins, S reduced them significantly greater than P did. Only simvastatin significantly increased apo A-I and significantly decreased apo C-II. Though atherogenic indices such as the (TC-HDL-C)/HDL-C and apo B/apo A-I ratios, as well as the LDL-C/apo B ratio, were significantly reduced by both statins, S reduced these atherogenic indices significantly greater than P did. S significantly increased the apo A-I/apo A-II ratio and significantly decreased the apo E/(apo C-II+apo C-III) ratio. To determine the mechanism by which simvastatin increased HDL-C, the relationship between changes in HDL-C and various parameters were examined. Changes in HDL-C were found to be negatively correlated with baseline HDL-C levels, changes in VLDL-C/LDL-C, and changes in LDL-TG. However there were no correlations of changes in HDL-C with baseline TG, changes in VLDL-TG or changes in LDL-C. Changes in HDL-C also showed positive correlations with changes in apo A-I, the HDL-C/apo A-I ratio, and the apo A-I/apo A-II ratio, respectively. These results suggest that simvastatin should be capable of promoting VLDL catabolism aside form the enhanced clearance of LDL from the circulation. The results also indicate that during these processes, except for patients with extremely high serum HDL-C levels, the number of HDL particles might increase partly as a result of increased VLDL hydrolysis and partly as a result of other unknown mechanisms. The precise mechanism by which HDL is increased by S remains to be elucidated.

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